Shrivastava et al Tropical Journal of Pharmaceutical Research, June 2009; 8 (3): 221-229 © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved . Available online at http://www.tjpr.org Research Article Flurbiprofen- and Suprofen-Dextran Conjugates: Synthesis, Characterization and Biological Evaluation Sushant K Shrivastava 1*, DK Jain 2, Prabhat K Shrivastava 1, and Piyush Trivedi 3 1Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, U.P., 2Department of Pharmacy, IPS Academy, Indore, M.P., 3School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, M.P., India Abstract Purpose: To synthesize and characterize the dextran conjugates of suprofen and flurbiprofen, and also evaluate their biological activities. Methods: Suprofen and flurbiprofen were individually reacted with carbonyldiimidazole to form acylimidazole, which, in turn, was reacted with the dextran of varying molecular weight (40 000, 60 000, and 110 000) to form drug-dextran conjugates. The structures of the synthesized dextran conjugates were confirmed by IR and NMR spectroscopy. In vitro hydrolysis of the conjugates were studied in buffer solutions (pH 7.4 and 9.0) and 80% human plasma (pH 7.4). The analgesic and antipyretic activities, as well as the ulcerogenic index of the conjugates were also evaluated in albino rats. Results: The mean degree of substitution of flurbiprofen and suprofen was between 8.0 to 9.5 % and 7.5 to 9.0 %, respectively. In vitro hydrolysis studies on the conjugates indicate faster hydrolysis at pH 9.0 than in pH 7.4 buffer solution and 80% human plasma (pH 7.4) with the process following First order kinetics. The analgesic activity of flurbiprofen-dextran conjugate (FD-110) suprofen-dextran conjugate (SD-110) was 64.23 and 41.50% which compare well with those of their parent drugs - flurbiprofen (72.60%) and suprofen (44.30%). Similar findings were made in respect of the antipyretic activity. Both flurbiprofen and suprofen showed deep ulceration, swelling and high intensity perforation in the gastric mucosa after seven days administration of flurbiprofen and suprofen with the ulcerogenic indices of 29.69 and 31.0 respectively, cpmpare with 5.88 and 6.06 for FD-110 and SD-110, respectively. Conclusion: Dextran can be employed as a pro-moiety or carrier for the delivery of flurbiprofen and suprofen and showed comparable analgesic and antipyretic activities with the parent drugs but with lower ulcerogenic indices. Keywords: Flurbiprofen, Suprofen, Dextran conjugates, In vitro hydrolysis, Analgesic-antipyretic activity Received: 9 Oct 2008 Revised accepted: 15 Dec 2008 *Corresponding author: Email: [email protected]; Phone: +91-543-2307049, Fax+91-542-2368428 Trop J Pharm Res, June 2009; 8 (3): 221 Shrivastava et al INTRODUCTION MATERIALS AND METHODS Not too long ago, polymeric prodrug Materials and equipment conjugates were ushered into the era of polymeric drug delivery. The task of obtaining Flurbiprofen and suprofen powders were obtained as gifts from Cipla Ltd, Mumbai, a versatile polymer as an ideal candidate in India. Dextran (molecular weight - 40 000, 60 drug delivery can be intricate since it has to 000 and 1 10 000) and N, N 1– 1 surmount several vigorous clinical barriers . carbonyldiimidazole (CDI) were purchased Dextran has excellent physicochemical from Sigma-Aldrich Chemicals Ltd, USA. properties and physiological acceptance such Silica gel G for TLC and solvents for HPLC as the capacity to be stored in depots, unique analysis were purchased from Merck India. All pharmacokinetic profiles, potential body other solvents and chemicals were of reagent 2,3 grade and obtained from Qualinges fine distribution and pharmacological efficacy . chemicals, Mumbai. The literature reveals that in most of the macromolecular or polymeric prodrug Ultra-violet spectra of the synthesized approaches, the drug is either linked by conjugates were generated using Shimadzu physical entrapment or by chemical linkage to 160-A, UV/Visible Spectrophotometer. I n vitro polymeric carriers 4-10 . hydrolysis of SD and FD conjugates was performed using a water-HPLC system Nonsteroidal anti-inflammatory drugs are (Rexdale, Canada) consisting of a model 6000A pump, a 710 B WISP auto injector, and among the most frequently used groups of a 490 multiple-wave length UV detector drugs to treat the disorders caused by 11-13 operated at ambient temperature. Separation inflammation . The phenyl propionic acid was carried out on octadecyl-bonded silica derivatives such as flurbiprofen {2-(3-fluoro-4- (5 µm, ODS-3) stainless steel column (10 x4.6 phenyl-phenyl) propanoic acid} and suprofen mm, i.d.) along with a 5 cm guard column of {2-(4-(thiophene-2-carbonyl) phenyl) the same material and particle size 10 µm. propanoic acid} possess analgesic and The mobile phase composition for separation antipyretic actions in addition to anti- was acetonitrile: water: phosphoric acid in the inflammatory action 14-18 . Unfortunately, ratio 45:54:1 v/v and acetonitrile: 0.67 M KH PO : triethylamine in the ratio 35: 65: 0.02 however, they also possess certain 2 4 v/v, respectively, for SD and FD conjugates. undesirable gastro-intestinal (GI) side effects The flow rate was maintained at 1.0 ml/min. such as gastric ulceration and hemorrhage which are attributed in part to the presence of The infrared spectra were recorded on an acidic group, and this can be masked Shimadzu 8300 FT-IR spectrophotometer temporarily by conjugating with a biopolymer using KBr pellets in the range 4000 to 400/cm. 1 dextran. Previously, we synthesized and H NMR spectra were obtained on a Bruker DRX -NMR spectrophotometer operating at a evaluated dextran conjugates of flurbiprofen frequency of 300 MH in DMSO-d . and suprofen for their anti-inflammatory Z 6 19,20 action . In this study, the dextran Synthesis of dextran conjugates conjugates of flurbiprofen (FD) and suprofen (SD) were prepared with the intention of Dextran conjugates of flubriprofen and achieving reductions of GI side-effects while suprofen were prepared by first activating the retaining their analgesic and antipyretic carboxylic group using CDI to obtain activities. flubriprofen and suprofen acylimidazole (FAI and SAI) 21 which were then condensed with Trop J Pharm Res, June 2009; 8 (3): 222 Shrivastava et al dextran of different molecular weight (40 000, absorption maxima of 248.4 nm and 296 nm 22,23 60 000, and 110 000) in situ to get FD-40, FD- respectively . In vitro hydrolysis of dextran 60 , FD-110 and SD-40, SD-60 , SD-110, conjugates was determined by HPLC using respectively, as shown in Scheme 1. The the mobile phase indicated earlier. The progress of the reaction was monitored by thin amount of hydrolyzed dextran conjugates was layer chromatography, which was performed derived from the measurement of the peak on silica gel (Merck No. 5554) as stationary area in relation to those of the standard drug phase and chloroform: methanol (7:3) as response under same conditions. mobile phase. N, N – carbonyl diimidazole (CDI) is moisture-sensitive and, therefore, dry Molecular weight determination solvents were used throughout and anhydrous conditions were maintained during the The molecular weight of the conjugates was experiment. determined by viscometery using Mark- Houwink Sakurada equation 24 ., which The IR and NMR spectral data of FD correlates molecular weight and intrinsic conjugates; IR (KBr, νmax cm -1): 1728.7 viscosity. (C=O str.), 2963 (aromatic str.), 736 (C-H [η] = k M α ……………….………. (1) aromatic bending), 3400-3278 (-OH str.of polymeric -OH dextran), 1568.(str. of biphenyl where [ η] is the intrinsic viscosity, M is the 1 ring), 1010 (C-F str.) H NMR (DMSO d 6, molecular weight, and k and α are constants ppm): 7.27- 7.52 (m, 8H, aromatic ring), 3.89 having values 7.24×10 -4 (dl/g) and 0.52 (q, 2H, -CH 2), 1.46 (t, 3H, -CH 3), 5.30-3.63 respectively. The values were determined by (m, anomeric protons of glucosidic ring), 2.0- plotting ηsp / C against C. Molecular weight 2.49 (-OH of dextran monomer). The IR and was then calculated using the above equation. NMR spectral data of SD conjugates; IR (KBr, νmax cm -1): 1723.6 (C=O str.), 2918 In vitro hydrolysis (aromatic str.), 742 (C-H aromatic bending), 3400-3243 (-OH str.of polymeric -OH In-vitro hydrolysis of the dextran conjugates dextran), 2970 (C-H str. of alkene), 1025 was studied in different phosphate buffer (thienyl str.), 1350 (C-S str.) 1H NMR (DMSO solutions (pH 7.4 and 9.0 and 80% human d6, ppm): 7.6- 8.0 (m, 4H, aromatic ring), 7.29- plasma pH 7.4) and the rate of hydrolysis of 7.55 (m, 3H, thiophene ring), 3.94 (q, 2H, - the dextran conjugates was computed as the CH 2), 1.23 (t, 3H, -CH 3), 5.32-3.44 (anomeric percent drug hydrolysed based on the protons of glucosidic ring), 2.0-2.5 (-OH of cumulative amount of drug hydrolysed divided dextran monomer) by the total amount of drug contained in the conjugate. The rate of hydrolysis and half-life Degree of substitution of the prepared conjugate were calculated. .2 303 a The degree of substitution of flurbiprofen and k = × …….………… (2) t a − x suprofen was determined by dissolving 20 mg .0 693 of the dextran conjugate in 20 ml solution of ()t1 / 2 = ……….…………….. (3) phosphate buffer (pH 9.0). The reaction k ° mixture was maintained at 70 C for one hour where k is the rate constant, t is the time in and left for 24 h for complete hydrolysis. It hours, a is the initial concentration of was then neutralized with 1N HCl. The conjugate, x is the amount of the conjugate amount of flurbiprofen and suprofen drugs hydrolyzed into the free drug, a-x is the released due to hydrolysis was extracted with amount of drug remaining in conjugated form chloroform and determined by HPLC at the and t ½ is the half life of conjugate.